Halogen-substituted amino dicarboxylic acid derivatives as medicaments for treating cardiovascular diseases

ABSTRACT

The invention relates to compounds of formula (I), to methods for their production and to the use thereof as medicaments.

[0001] The present invention relates to novel halogen-substitutedaminocarboxylic acid derivatives which stimulate soluble guanylatecyclase also via a novel mechanism of action which takes place withoutinvolvement of the heme group of the enzyme, to their preparation and totheir use as medicaments, in particular as medicaments for treatingcardiovascular disorders.

[0002] One of the most important cellular transmission systems inmammalian cells is cyclic guanosine monophosphate (cGMP). Together withnitrogen monoxide (NO), which is released from the endothelium andtransmits hormonal and mechanical signals, it forms the NO/cGMP system.Guanylate cyclases catalyze the biosynthesis of cGMP from guanosinetriphosphate (GTP). The representatives of this family disclosed to datecan be divided both according to structural features and according tothe type of ligands into two groups: the particulate guanylate cyclaseswhich can be stimulated by natriuretic peptides, and the solubleguanylate cyclases which can be stimulated by NO. The soluble guanylatecyclases consist of two subunits and very probably contain one heme perheterodimer, which is part of the regulatory center. The latter is ofcentral importance for the mechanism of activation. NO is able to bindto the iron atom of heme and thus markedly increase the activity of theenzyme. Heme-free preparations cannot, by contrast, be stimulated by NO.CO is also able to attach to the central iron atom of heme, but thestimulation by CO is distinctly less than that by NO.

[0003] Through the production of cGMP and the regulation, resultingtherefrom, of phosphodiesterases, ion channels and protein kinases,guanylate cyclase plays a crucial part in various physiologicalprocesses, in particular in the relaxation and proliferation of smoothmuscle cells, in platelet aggregation and adhesion and in the neuronalsignal transmission, and in disorders caused by an impairment of theaforementioned processes. Under pathophysiological conditions, theNO/cGMP system may be suppressed, which may lead for example to highblood pressure, platelet activation, increased cell proliferation,endothelial dysfunction, atherosclerosis, angina pectoris, heartfailure, thromboses, stroke and myocardial infarction.

[0004] A possible way of treating such disorders which is independent ofNO and aims at influencing the cGMP signal pathway in organisms is apromising approach because of the high efficiency and few side effectswhich are to be expected.

[0005] Compounds, such as organic nitrates, whose effect is based on NOhave to date been exclusively used for the therapeutic stimulation ofsoluble guanylate cyclase. NO is produced by bioconversion and activatessoluble guanylate cyclase by attaching to the central iron atom of heme.Besides the side effects, the development of tolerance is one of thecrucial disadvantages of this mode of treatment.

[0006] Some substances which directly stimulate soluble guanylatecyclase, i.e. without previous release of NO, have been described inrecent years, such as, for example,3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1, Wu et al., Blood84 (1994), 4226; Mülsch et al., Br. J. Pharmacol. 120 (1997), 681),fatty acids (Goldberg et al, J. Biol. Chem. 252 (1977), 1279),diphenyliodonium hexafluorophosphate (Pettibone et al., Eur. J.Pharmcol. 116 (1985), 307), isoliquiritigenin (Yu et al., Brit. J.Pharmacol. 114 (1995), 1587) and various substituted pyrazolederivatives (WO 98/16223, WO 98/16507 and WO 98/23619).

[0007] The stimulators of soluble guanylate cyclase described abovestimulate the enzyme either directly via the heme group (carbonmonoxide, nitrogen monoxide or diphenyliodonium hexafluorophosphate) byinteraction with the central iron of the heme group and a resultingchange in conformation which leads to an increase in enzyme activity(Gerzer et al., FEBS Lett. 132(1981), 71), or via a heme-dependentmechanism which is independent of NO but leads to a potentiation of thestimulating action of NO or CO (for example YC-1, Hoenicka et al., J.Mol. Med. (1999) 14; or the pyrazole derivatives described in WO98/16223, WO 98/16507 and WO 98/23619).

[0008] The stimulating action of isoliquiritigenin and of fatty acids,such as, for example, arachidonic acid, prostaglandin endoperoxides andfatty acid hydroperoxides, on soluble guanylate cyclase claimed in theliterature could not be confirmed (cf., for example, Hoenicka et al., J.Mol. Med. 77 (1999), 14).

[0009] If the heme group is removed from soluble guanylate cyclase, theenzyme still has detectable catalytic basal activity, i.e. cGMP is stillbeing formed. The residual catalytic basal activity of the heme-freeenzyme cannot be stimulated by any of the known stimulators mentionedabove.

[0010] Stimulation of heme-free soluble guanylate cyclase byprotoporphyrin IX has been described (Ignarro et al., Adv. Pharmacol. 26(1994), 35). However, protoporphyrin IX can be considered to be a mimicof the NO-heme adduct, as a consquence of which the addition ofprotoporphyrin IX to soluble guanylate cyclase would be expected toresult in the formation of a structure of the enzyme corresponding toheme-containing soluble guanylate cyclase stimulated by NO. This is alsoconfirmed by the fact that the stimulating action of protoporphyrin IXis increased by the above-described NO-independent but heme-dependentstimulator YC-1 (Mülsch et al., Naunyn Schmiedebergs Arch. Pharmacol.355, R47).

[0011] In contrast to the above-described compounds, known from theprior art as stimulators of soluble guanylate cyclase, the compoundsaccording to the invention are capable of stimulating both theheme-containing and the heme-free form of soluble guanylate cyclase.Thus, in the case of these novel stimulators, stimulation of the enzymeis effected via a heme-independent path, and this is also confirmed bythe fact that firstly the novel stimulators do not have any synergisticaction with NO at the heme-containing enzyme and that secondly theaction of these novel stimulators cannot be blocked by theheme-dependent inhibitor of soluble guanylate cyclase, i.e.1H-1,2,4-oxadiazole-(4,3a)-quinoxalin-1-one (ODQ).

[0012] This is a novel therapeutic approach for treating cardiovasculardisorders and other disorders accessible to therapy by influencing thecGMP signal pathway in organisms.

[0013] EP-A-0 345 068 describes, inter alia, the aminoalkanecarboxylicacid (1) as an intermediate in the synthesis of GABA antagonists:

[0014] WO 93/00359 describes the aminoalkanecarboxylic acid (2) as anintermediate in peptide synthesis and its use as active compound fortreating disorders of the central nervous system:

[0015] However, neither of these two publications describes that suchaminoalkanecarboxylic acids may have a stimulating effect, independentof the heme group present in the enzyme, on soluble guanylate cyclase.

[0016] Substances having a structure similar to that of the compoundsaccording to the invention are furthermore known from WO 01/19776, WO01/19355, WO 01/19780 and WO 01/19778.

[0017] The present invention relates to compounds of the general formula(I)

[0018] where

[0019] R¹ is located in the meta- or para-position to the ethylaminoradical and represents halogen;

[0020] R² represents H or halogen;

[0021] R³ represents H or halogen;

[0022] R⁴ represents C₃₋₈-cycloalkyl or phenyl, where the phenyl radicalmay additionally carry a substituent from the group consisting ofhalogen, CN, OMe, CF₃;

[0023] with the proviso that R⁴ may not represent phenyl which carries asubstituent CF₃ or OMe in the para-position to the point of attachmentif, simultaneously, R¹ is in the meta-position to the ethylamino radicaland represents F and R² and R³ each represent H;

[0024] and their salts, isomers and hydrates.

[0025] According to a preferred embodiment, the present inventionrelates to compounds of the formula (I) where

[0026] R¹ is located in the meta- or para-position to the ethylaminoradical and represents F, Cl or Br;

[0027] R² represents H or F or Cl;

[0028] R³ represents H;

[0029] R⁴ represents cyclohexyl or phenyl, where the phenyl radical mayadditionally carry a substituent from the group consisting of F, Cl, Br,CN, OMe, CF₃;

[0030] with the proviso that R⁴ may not represent phenyl which carries asubstituent CF₃ or OMe in the para-position to the point of attachmentif, simultaneously, R¹ is in the meta-position to the ethylamino radicaland represents F and R² and R³ each represent H;

[0031] and their salts, isomers and hydrates.

[0032] According to a particularly preferred embodiment, the presentinvention relates to compounds of the formula (I) where

[0033] R¹ is located in the meta-position to the ethylamino radical andrepresents Cl;

[0034] R² represents H or Cl;

[0035] R³ represents H;

[0036] R⁴ represents cyclohexyl or phenyl, where the phenyl radical mayadditionally carry a substituent from the group consisting of F, Cl, CN,OMe, CF₃;

[0037] and their salts, isomers and hydrates.

[0038] According to a further particularly preferred embodiment, thepresent invention relates to compounds of the formula (I) in which

[0039] R¹ is located in the para-position to the ethylamino radical andrepresents Cl or F;

[0040] R² represents H or Cl;

[0041] R³ represents H;

[0042] R⁴ represents cyclohexyl or phenyl, where the phenyl radical mayadditionally carry a substituent from the group consisting of F, Cl, CN,OMe, CF₃;

[0043] and their salts, isomers and hydrates.

[0044] The compounds according to the invention of the general formula(I) may also be in the form of their salts. Mention may generally bemade here of salts-with organic or inorganic bases or acids.

[0045] Physiologically acceptable salts are preferred for the purposesof the present invention. Physiologically acceptable salts of thecompounds according to the invention may be salts of the substancesaccording to the invention with mineral acids, carboxylic acids orsulfonic acids. Particularly preferred examples are salts withhydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionicacid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acidor benzoic acid.

[0046] Physiologically acceptable salts may likewise be metal orammonium salts of the compounds according to the invention having a freecarboxyl group. Particularly preferred examples are sodium, potassium,magnesium or calcium salts, and ammonium salts derived from ammonia, ororganic amines, such as, for example, ethylamine, di- or triethylamine,di- or triethanolamine, dicyclohexylamine, dimethylaminoethanol,arginine, lysine or ethylenediamine.

[0047] The compounds according to the invention may exist instereoisomeric forms which are either like image and mirror image(enantiomers), or not like image and mirror image (diastereomers). Theinvention relates both to the enantiomers or diastereomers and to theirrespective mixtures. The racemic forms, like the diastereomers, can beseparated into the stereoisomerically uniform components in a knownmanner, for example by optical resolution or chromatographic separation.Double bonds present in the compounds according to the invention can bein the cis or trans configuration (Z or E form).

[0048] For the purposes of the present invention, the substituents are,unless defined otherwise, generally as defined below:

[0049] Alkyl generally represents a straight-chain or branchedhydrocarbon radical having 1 to 20 carbon atoms. Examples which may bementioned are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl,isopentyl, hexyl, isohexyl, heptyl, isoheptyl, octyl and isooctyl,nonyl, decyl, dodeyl, eicosyl.

[0050] Alkoxy generally represents a straight-chain or branchedhydrocarbon radical having 1 to 14 carbon atoms which is attached via anoxygen atom. Examples which may be mentioned are methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, pentoxy isopentoxy, hexoxy,isohexoxy, heptoxy, isoheptoxy, octoxy or isooctoxy. The terms “alkoxy”and “alkyloxy” are used synonymously.

[0051] Cycloalkyl generally represents a cyclic hydrocarbon radicalhaving 3 to 8 carbon atoms. Preference is given to cyclopropyl,cyclopentyl and cyclohexyl. Cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl may be mentioned by way of example.

[0052] Halogen, for the purposes of the invention, represents fluorine,chlorine, bromine and iodine.

[0053] The present invention furthermore relates to a process forpreparing the compounds of the formula (I), characterized in that

[0054] compounds of the formula (II)

[0055] in which

[0056] R¹, R², R³ and R⁴ are as defined above,

[0057] are reacted with a C₁₋₆-alkyl 4-formylbenzoate in an organicsolvent with heating and with simultaneous or subsequent addition of areducing agent to give compounds of the formula (III)

[0058] where

[0059] R¹, R², R³ and R⁴ are as defined above and Q represents aC₁₋₆-alkyl radical,

[0060] then reacted with a C₁₋₆-alkyl ω-halovalerate in an organicsolvent in the presence of a base with heating to give compounds of theformula (IV)

[0061] where

[0062] R¹, R², R³ and R⁴ and Q are as defined above and Q′ represents aC₁₋₆-alkyl radical,

[0063] and the compounds of the formula (IV) are then hydrolyzed underalkaline conditions to give the compounds of the formula (I).

[0064] Bases which are preferred for the processes according to theinvention include basic compounds which are customarily used for basicreactions. Preference is given to using alkali metal hydrides, such as,for example, sodium hydride or potassium hydride, or alkali metalalkoxides, such as sodium methoxide, sodium ethoxide, potassiummethoxide, potassium ethoxide or potassium t-butoxide, or carbonates,such as sodium carbonate, cesium carbonate or potassium carbonate, oramides, such as sodium amide or lithium diisopropylamide, ororganolithium compounds, such as phenyllithium, butyllithium ormethyllithium, or sodium hexamethyldisilazane.

[0065] Solvents which are preferred for converting the compounds of theformula (II) into the compounds of the formula (III) are customaryorganic solvents which do not change under the reaction conditions.Preference is given to using, for the process according to theinvention, ethers, such as diethyl ether, butyl methyl ether, dioxane,tetrahydrofuran, glycol dimethyl ether or diethylene glycol dimethylether, or hydrocarbons, such as benzene, toluene, xylene or petroleumether, or alcohols, such as methanol or ethanol, or halogenatedhydrocarbons, such as carbon tetrachloride, chloromethane ordichloromethane. It is, of course, also possible to use mixtures of thesolvents mentioned above. Preference according to the invention is givento using toluene and/or methanol.

[0066] The compounds of the formula (II) are reacted initially with aC₁₋₆-alkyl 4-formylbenzoate giving a Schiff base which is then reducedwith customary reducing agents, such as, for example, NaBH₄, H₂/Pd/C,etc., or reacted directly under the conditions of a reductive alkylationin the presence of a reducing agent, such as, for example, H₂/Pd/C,NaCNBH₃, NaH(OAc)₃ (cf. Patai, Ed., The Chemistry of the Carbon-NitrogenDouble Bond, pp. 276-293 and the literature cited therein). Depending onthe nature of the starting material, the reaction can be carried out atroom temperature or requires heating at 50-110° C. for a number of hoursto a number of days. The reaction can be carried out at atmosphericpressure, elevated or reduced pressure (for example in a range of from0.5 to 5 bar). In general, the reaction is carried out at atmosphericpressure. C₁₋₆-Alkyl 4-formylbenzoates are commercially available, knownfrom the literature, or they can be synthesized analogously to processesknown from the literature (cf., for example, J. Med. Chem. 1989, 32,1277; Chem. Ber. 1938, 71, 335; Bull. Soc. Chim. Fr. 1996, 123, 679,WO96/11902; DE-2209128; Synthesis 1995, 1135; Bull. Chem. Soc. Jpn.1985, 58, 2192, Synthesis 1983, 942; J. Am. Chem. Soc. 1992, 114, 8158).

[0067] The conversion of the compounds of the formula (III) into thecompounds of the formula (IV) can preferably be carried out inacetonitrile or butyronitrile, in each case in the presence of a base,such as sodium carbonate, Et₃N, DABCO, K₂CO₃, KOH, NaOH or NaH. Ingeneral, the reaction can be carried out in a temperature range of from−20° C. to +90° C., preferably from 0° C. to +70° C. The reaction can becarried out under atmospheric pressure, elevated or reduced pressure(for example in a range of from 0.5 to 5 bar). In general, the reactionis carried out under atmospheric pressure. However, suitable solventsare, in principle, the solvents mentioned above for the conversion ofthe compounds of the formula (II) into the compounds of the formula(II). According to the invention, the alkyl ω-halovalerate used is,preferably, the corresponding methyl 1-bromovalerate. Alkylω-halovalerates are commercially available, known from the literature orcan be synthesized according to processes known from the literature(cf., for example, J. Chem. Soc. 1958, 3065).

[0068] The compounds of the formula (IV) are then converted into thecompounds of the formula (I) by hydrolysis of the ester functions to thefree carboxyl groups, for example by adding aqueous solutions of strongacids, such as, for example, HCl or H₂SO₄, or strong bases, such as, forexample, NaOH, KOH or LiOH. The reaction can be carried out in one ofthe organic solvents mentioned above, in water or in mixtures of organicsolvents or in mixtures of organic solvents with water. Preferenceaccording to the invention is given, for example, to carrying out thereaction in a mixture of water and methanol or dioxane. In general, thereaction can be carried out in a temperature range of from −20° C. to+90° C., preferably from 0° C. to +90° C. The reaction can be carriedout under atmospheric pressure, elevated or reduced pressure (forexample in a range of from 0.5 to 5 bar). In general, the reaction iscarried out under atmospheric pressure.

[0069] If the compounds of the formula (II) are not commerciallyavailable, they can be obtained in the manner described below. 4- or5-halo-2-hydroxybenzaldehydes, which are commercially available or knownfrom the literature, give, by reaction with appropriate benzyl halides,preferably benzyl chlorides, substituted in the 4-position in an organicsolvent, such as acetonitrile, in the presence of a base, such as, forexample, potassium carbonate, reaction of the aldehyde group withnitromethane in an organic solvent, such as, for example, ethanol, inthe presence of a base, such as, for example, methylamine, and reductionof the resulting nitroethenyl function in two steps, initially with analkali metal hydride, such as, for example, LiAl₄, to give thecorresponding hydroxylamine ethyl function, and then with Zn in acidicmedium, such as, for example, in the presence of acetic acid, thecorresponding amine of the formula (II).

[0070] The compounds of the general formula (I) according to theinvention show a valuable range of pharmacological effects which couldnot have been predicted.

[0071] The compounds of the general formula (I) according to theinvention bring about vasorelaxation and an inhibition of plateletaggregation and lead to a reduction in blood pressure and an increase inthe coronary blood flow. These effects are mediated by directstimulation of soluble guanylate cyclase and an intracellular increasein cGMP.

[0072] They can therefore be employed in medicaments for the treatmentof cardiovascular disorders such as, for example, for the treatment ofhigh blood pressure and heart failure, stable and unstable anginapectoris, peripheral and cardiac vascular disorders, of arrhythmias, forthe treatment of thromboembolic disorders and ischemias such asmyocardial infarction, stroke, transitory and ischemic attacks,disturbances of peripheral blood flow, prevention of restenosis such asafter thrombolysis therapies, percutaneous transluminal angioplasties(PTAs), percutaneous transluminal coronary angioplasties (PTCAs), bypassand for the treatment of arteriosclerosis, fibrotic disorders, such asfibrosis of the liver or pulmonary fibrosis, asthmatic disorders anddiseases of the urogenital system such as, for example, prostatehypertrophy, erectile dysfunction, female sexual dysfunction andincontinence and also for the treatment of glaucoma.

[0073] The compounds of the general formula (I) described in the presentinvention are also active compounds suitable for controlling centralnervous system diseases characterized by disturbances of the NO/cGMPsystem. They are suitable in particular for removing cognitive deficits,for improving learning and memory performances and for treatingAlzheimer's disease. They are also suitable for treating disorders ofthe central nervous system such as states of anxiety, tension anddepression, CNS-related sexual dysfunctions and sleep disturbances, andfor controlling pathological disturbances of the intake of food,stimulants and addictive substances.

[0074] The active compounds are furthermore also suitable for regulatingcerebral blood flow and thus represent effective agents for controllingmigraine. They are also suitable for the prophylaxis and control of thesequelae of cerebral infarction (apoplexia cerebri) such as stroke,cerebral ischemias and craniocerebral trauma. The compounds of thegeneral formula (I) according to the invention can likewise be employedfor controlling states of pain.

[0075] In addition, the compounds according to the invention have ananti-inflammatory effect and can therefore be employed asanti-inflammatory agents.

[0076] As a particular and surprising feature, the compounds of thepresent invention have an unexpectedly long duration of action.

[0077] Vasorelaxant Effect In Vitro

[0078] Rabbits are anesthetized or killed by intravenous injection ofthiopental sodium (about 50 mg/kg) and exsanguinated. The arteriasaphena is removed and divided into rings 3 mm wide. The individualrings are in each case mounted on a pair of hooks of triangular shape,open at the ends and made of special wire (Remanium®) having a diameterof 0.3 mm. Under pretension, each ring is introduced into a 5 ml organbath containing carbogen-gassed Krebs-Henseleit solution at 37° C. withthe following composition (mM): NaCl: 0.119; KCl: 4.8; CaCl₂×2H₂O: 1;MgSO₄×7H₂O: 1.4; KH₂PO₄: 1.2; NaHCO₃: 25; glucose: 10; bovine serumalbumin: 0.001%. The force of contraction is detected with Statham UC2cells, amplified and digitized via A/D converters (DAS-1802 HC, KeithleyInstruments, Munich) and recorded in parallel on chart recorders.Contractions are generated by adding phenylephrine.

[0079] After several (generally 4) control cycles, the substance to beinvestigated is added in each further run in increasing dosage, and theheight of the contraction reached under the influence of the testsubstance is compared with the height of the contraction reached in thelast preceding run. The concentration necessary to reduce the height ofthe control value by 50% (IC₅₀) is calculated from this. The standardapplication volume is 5 μl. The DMSO content in the bath solutioncorresponds to 0.1%.

[0080] The results are shown in Table 1: TABLE 1 Vasorelaxant effect invitro Example IC₅₀ (nM) 1 1.1 2 1.5 3 1 6 8.1 9 8.3

[0081] Stimulation of Recombinant Soluble Guanylate Cyclase (sGC) InVitro

[0082] The investigations of the stimulation of recombinant solubleguanylate cyclase (sGC) and the compounds according to the inventionwith and without sodium nitroprusside and with and without theheme-dependent sGC inhibitor 1H-1,2,4-oxadiazole-(4,3a)-quinoxalin-1-one(ODQ) were carried out according to the method described in detail inthe following literature reference: M. Hoenicka, E. M. Becker, H.Apeler, T. Sirichoke, H. Schroeder, R. Gerzer and J.-P. Stasch: Purifiedsoluble guanylyl cyclase expressed in a baculovirus/Sf9 system:stimulation by YC-1, nitric oxide, and carbon oxide. J. Mol. Med. 77(1999): 14-23.

[0083] The heme-free guanylate cyclase was obtained by adding Tween 20to the sample buffer (final concentration 0.5%).

[0084] Activation of sGC by a test substance is stated as n-foldstimulation of basal activity.

[0085] Investigation of the Antifibrotic Action of the Substances InVivo

[0086] Method

[0087] The antifibrotic action of the substances was investigated usingthe model of the porcine serum-induced rat liver fibrosis. Treatmentwith heterologous serum, for example porcine serum in rats, is a methodfrequently used in the literature for inducing fibrosis of the liverwith subsequent cirrhosis which, in contrast to other models, causesonly minimal damage and inflammation of the parenchyma cells of theliver (Bhunchet, E. and Wake, K. (1992): Role of mesenchymal cellpopulations in porcine serum-induced rat liver fibrosis. Hepatology 16:1452-1473). Female Sprague Dawley rats were treated 2× per week with 0.5ml/animal of sterile porcine serum (Sigma) i.p., control animals weretreated with sterile physiological saline (2× per week 0.5-m/animali.p.). The treatment with test substance (1× per day in 5 mvkg of p.o.solvent comprising 20% Cremophor, 10% Transcutol and 70% H₂O) wascarried out in parallel to the treatment with porcine serum. After sevenweeks of treatment, the animals were killed and the livers were removedin order to quantify the collagen content.

[0088] For the histological examination of the liver tissue,standardized transverse tissue cylinders (about 10×2 mm) were punchedout of the right anterior lobe of the liver.

[0089] For the detection of scar collagen caused by liver fibrosis,frozen sections were stained with 0.1% strength Pikrosirius Redsolution.

[0090] Fast Green was used as counterstain to enhance contrasts. In eachsection, the extent of liver fibrosis was determined as a percentage ofthe area stained by Pikrosirius Red of the total area measured. Theparameters of the video microscopic stain detection were standardizedand kept constant for the entire experiment. 64 fields of a standardizedgrid of 31 mm² were measured using a final amplification of 100. Forsemiautomatic morphometry, a Leica Quantimed 500MC (Leica Germany) wasused.

[0091] To determine OH-proline according to Prockop and Udenfried(Prockop, D. J. and Udenfried, S. A. (1960): A specific method for theanalysis of hydroxyproline in tissues and urine. Anal. Biochem. 1:228-239), in each case 50-100 mg of liver tissue were dried and boiledwith 6N HCl for about 17 hours. The acid was evaporated in a vacuumdrying cabinet and the residue was then dissolved in 5 ml of distilledwater and filtered. 200 μl of the filtered solution were incubated atroom temperature with 200 μl of ethanol and 200 μl of oxidation solution(7% strength aqueous chloramine T hydrate solution, diluted 1:4 withacetate/citrate buffer pH 6.0) for 25 min. 400 μl of Ehrlich's reagent(12 g of 4-dimethylaminobenzaldehyde in 20 ml of ethanol+2.74 ml ofconcentrated sulfuric acid in 20 ml of ethanol) were then added. After 3hours of incubation at 35° C., absorption at 573 nm was measured.Aqueous OH-proline solutions (Sigma) were used for the calibrationcurve. The OH-proline content of the liver samples was calculated in mgper g of liver dry weight.

[0092] Results

[0093] The OH-proline values agree very well with the results of themorphometric fibrosis measurement: without simultaneous administrationof substance, the porcine serum treatment results in a pronouncedaccumulation of collagen in the liver. The formation of these collagendeposits is reduced by treatment with the substances in a dose-dependentmanner.

[0094] The present invention includes pharmaceutical preparations which,in addition to non-toxic, inert, pharmaceutically acceptable carriers,comprises the compounds according to the invention, in particular thecompounds of the general formula (I), and processes for preparing thesepreparations.

[0095] The active compound, if appropriate in one or more of thecarriers listed above, can also be present in microencapsulated form.

[0096] The therapeutically effective compounds, in particular thecompounds of the general formula (I), should be present in thepharmaceutical preparations detailed above in a concentration of about0.1 to 99.5, preferably of about 0.5 to 95, % by weight of the completemixture.

[0097] The pharmaceutical preparations detailed above may, apart fromthe compounds according to the invention, in particular the compounds ofthe general formula (I), also contain other active pharmaceuticalingredients.

[0098] It has generally proved to be advantageous both in human and inveterinary medicine to administer the active compound(s) according tothe invention in total amounts of about 0.5 to about 500, preferably 5to 100, mg/kg of body weight every 24 hours, where appropriate in theform of a plurality of single doses, to achieve the desired results. Asingle dose contains the active compound(s) according to the inventionpreferably in amounts of about 1 to about 80, in particular 3 to 30,mg/kg of body weight.

[0099] Below, the present invention is illustrated in more detail usingnon-limiting preferred examples. Unless indicated otherwise, allquantities refer to percent by weight.

EXAMPLES

[0100] Abbreviations: RT: room temperature EA: ethyl acetate BABA:n-butyl acetate/n-butanol/glacial acetic acid/phosphate buffer pH 6(50:9:25.15; org. phase)

[0101] Mobile Phases for Thin-Layer Chromatography: T1 E1: toluene-ethylacetate (1:1) T1 EtOH1: toluene-methanol (1:1) C1 E1: cyclohexane-ethylacetate (1:1) C1 E2: cyclohexane-ethyl acetate (1:2)

[0102] Starting Materials

Ex. I 2-[(4-cyclohexylbenzyl)oxy]-4-fluorobenzaldehyde

[0103]

[0104] 2 g (14.3 mmol) of 2-hydroxy-4-fluorobenzaldehyde (CAS 348-28-7)and 2.37 g (17.1 mmol) of anhydrous potassium carbonate are added to asolution of 3.13 g (15 mmol) of 4-cyclohexylbenzyl chloride (CAS4463-31-4) in 50 ml of dry acetonitrile, and the mixture is heated atreflux for six hours. The mixture is then concentrated to dryness usinga rotary evaporator, water and a little ether are added and theinsoluble product is isolated by filtration. This gives 4.4 g (14.1mmol, 99% yield) of a solid.

[0105] R_(f) (cyclohexane/ethyl acetate 1:1): 0.77.

[0106]¹H NMR (300 MHz, DMSO-d₆, δ/ppm): 10.29 (1H, s), 7.78 (1H, dd),7.41 (2H, d), 7.28-7.20 (3H, m), 6.93 (1H, dt), 5.26 (2H, s), 2.50 (1H,m, partially obscured by DMSO), 1.83-1.67 (5H, m), 1.47-1.18 (5H, m).

[0107] MS (DC₁, NH₃): 642 (2M+NH₄ ⁺), 502, 330 (M+NH₄ ⁺), 207.

Ex. II 2-[(4-cyclohexylbenzyl)oxy]-4-fluoro-1-[2-nitroethenyl]benzene

[0108]

[0109] A mixture of 460 mg of anhydrous sodium carbonate and 460 mg ofmethylamine hydrochloride in 5 ml of absolute ethanol is stirred for 15minutes and then filtered into a solution of 4.4 g (14.09 mmol) of2-[(4-cyclohexylbenzyl)oxy]4-fluorobenzaldehyde from Ex. I. 1.18 ml(21.69 mmol) of nitromethane and a spatula tip of sodium acetate areadded. The mixture is heated at 60° C. After a while, a yellow solidbegins to precipitate. After three hours, the reaction is ended. Themixture is cooled in an ice/water bath and the precipitated product isthen filtered off and washed with a little cold ethanol. This gives 3.65g (10.27 mmol, 73% yield) of a yellow solid.

[0110] Melting point: 86° C.

[0111] R_(f) (cyclohexane/dichloromethane 4:1): 0.64.

[0112]¹H-NMR (400 MHz, DMSO-d₆, δ/ppm): 8.18 (1H, d), 8.07 (1H, d), 7.92(1H, dd), 7.41 (2H, d), 7.29 (2H, d), 7.23 (1H, dd), 6.94 (1H, dt), 5.27(2H, s), 2.52 (1H, m, partially obscured by DMSO), 1.81-1.67 (5H, m),1.47-1.19 (5H, m).

[0113] MS (DCI, NH3): 390 (M+NH₃ ⁺NH₄ ⁺), 373 (M+NH₄ ⁺).

Ex. III: 2-{2-[(4-cyclohexylbenzyl)oxy]-4-fluorophenyl}ethylamine

[0114]

[0115] 30.5 ml (30.5 mmol) of a 1-molar solution of LiAIH₄ in THF isdiluted with a further 20 ml of anhydrous THF, and, at −78° C., asolution of 3.6 g (10.13 mmol) of2-[(4-cyclohexylbenzyl)oxy]-4-fluoro-1-[2-nitroethenyl]benzene from Ex.II in 30 ml of anhydrous THF is added dropwise. The mixture is stirredinitially at −78° C. for 30 minutes and then at 0° C. for 30 minutes. 50ml of aqueous sodium potassium tartrate solution are then addedcarefully, the mixtureis diluted with ether and the organic phase isseparated off. The organic phase is washed successively with water andsaturated sodium chloride solution, dried over anhydrous sodium sulfateand, after filtration, freed from the solvent. The resulting crudeproduct is purified by flash chromatography (silica gel,cyclohexane/ethyl acetate 1:1). This gives 1.25 g (3.64 mmol, 38% yield)ofN-(2-{2-[(4-chlorohexylbenzyl)oxy]-4-fluorophenyl}ethyl)hydroxylamine.

[0116] R_(f) (cyclohexane/ethyl acetate 1:1): 0.10.

[0117]¹H-NMR (200 MHz, DMSO-d₆, δ/ppm): 7.38 (2H, d), 7.27-7.12 (4H, m),6.92 (1H, dd), 6.68 (1H, dt), 5.61 (1H, s broad), 5.07 (2H, s),2.93-2.85 (2H, m), 2.78-2.68 (2H, m), 2.49 (1H, m, partially obscured byDMSO), 1.84-1.66 (5H, m), 1.49-1.27 (5H, m).

[0118] MS (DCI, NH₃): 344 (M+H⁺).

[0119] 1.05 g (3.06 mmol) ofN-(2-[2-[(4-cyclohexylbenzyl)oxy]4-fluorophenyl}ethyl)-hydroxylamine aredissolved in 10 ml of glacial acetic acid, and 1 g (15.29 mmol) of zincdust is added a little at a time. After 20 hours of stirring at roomtemperature, the mixture is stirred into an excess of saturated sodiumbicarbonate solution. The mixture is extracted with ethyl acetate. Thecombined extracts are washed successively with saturated sodiumbicarbonate solution, water and saturated sodium chloride solution.Drying over sodium sulfate. This gives 1.0 g (3.05 mmol, 99% yield) ofproduct.

[0120] R_(f) (ethyl acetate/methanol 7:3): 0.08.

[0121]¹H-NMR (400 MHz, DMSO-d₆, δ/ppm): 7.37 (2H, d), 7.23 (2H, d), 7.14(1H, dd), 6.92 (1H, dd), 6.68 (1H, dt), 5.06 (2H, s), 2.73-2.70 (2H, m),2.67-2.62 (2H, m), 2.49 (1H, m, partially obscured by DMSO), 1.90 (2H, sbroad), 1.80-1.68 (5H, m), 1.42-1.20 (5H, m).

[0122] MS (DCI, NH₃): 328 (M+H⁺).

Ex. IV Methyl4-{[(2-{2-[(4-cyclohexylbenzyl)oxy]-4-fluorophenyl}ethyl)-amino]methyl}benzoate

[0123]

[0124] 500 mg (1.53 mmol) of2-{2-[(4-cyclohexylbenzyl)oxy]4-fluorophenyl)ethylamine from Ex. III and251 mg (1.53 mmol) of methyl 4-formylbenzoate in 50 ml of toluene areboiled in a water separator for 30 minutes. The toluene is then removedusing a rotary evaporator and replaced by 15 ml of methanol. At 0° C.,58 mg (1.53 mmol) of NaBH₄ are added to the methanolic solution and themixture is then stirred at room temperature for 30 minutes. The mixtureis neutralized by addition of 5% strength aqueous sodium dihydrogenphosphate solution and diluted with ether and water, and the organicphase is separated off. After drying over Na₂SO₄ and evaporation of thesolvent, the product is purified by flash chromatography (silica gel,cyclohexane/ethyl acetate 1:1). This gives 436 mg (0.92 mmol, 60% yield)of a colorless oil.

[0125] R_(f) (cyclohexane/ethyl acetate 1:1): 0.17.

[0126]¹H-NMR (300 MHz, DMSO-d₄, δ/ppm): 7.87 (2H, d), 7.41 (2H, d), 7.29(2H, d), 7.19 (2H, d), 7.14 (1H, dd), 6.90 (1H, dd), 6.67 (1H, dt), 5.02(2H, s), 3.83 (3H, s), 3.76 (2H, s), 2.73-2.62 (4, m), 2.48 (1H, m,partially obscured by DMSO), 2.40 (1H, broad), 1.81-1.68 (5H, m),1.43-1.20 (5H, m).

[0127] MS (ESI): 476 (M+H⁺).

Ex. V Methyl4-{[(2-{2-[(4-cyclohexylbenzyl)oxy]-4-fluorophenyl}ethyl)(5-methoxy-5-oxo-pentyl)amino]methyl}benzoate

[0128]

[0129] 54 mg (0.50 mmol) of anhydrous sodium carbonate are added to asolution of 200 mg (0.42 mmol) of methyl4-{[(2-{2-[(4-cyclohexylbenzyl)oxy]4-fluorophenyl}ethyl)amino]methyl}benzoatefrom Ex. IV and 67 μl (0.50 mmol) of methyl 5-bromovalerate in 10 ml ofbutyronitrile, and the mixture is heated at reflux for 48 hours. Themixture is then concentrated, taken up in ethyl acetate and washed withwater. After drying over Na₂SO₄, filtration and concentration, theproduct is purified by flash chromatography (silica gel,cyclohexane/ethyl acetate 9:1). This gives 150 mg (0.25 mmol, 60% yield)of a colorless oil.

[0130] R_(f) (cyclohexane/ethyl acetate 1:1): 0.66.

[0131]¹H-NMR (300 MHz, DMSO-d₆, δ/ppm): 7.83 (2H, d), 7.33 (2H, d), 7.27(2H, d), 7.18 (2H, d), 7.12 (1H, dd), 6.90 (1H, dd), 6.66 (1H, dt), 4.97(2H, s), 3.83 (3H, s), 3.59 (2H, s), 3.54 (3H, s), 2.72-2.65 (2H, m),2.58-2.51 (2H, m), 2.48 (1H, m, partially obscured by DMSO), 2.39 (2H,t), 2.17 (2H, t), 1.80-1.67 (5H, m), 1.46-1.23 (9H, m).

[0132] MS (ESI): 590 (M+H⁺).

Synthesis Examples Ex. 14-{[(4-carboxybutyl)(2-{2-[(4-cyclohexylbenzyl)oxy]-4-fluorophenyl}ethyl)amino]methyl}BenzoicAcid

[0133]

[0134] 8 ml of a 2-molar solution of NaOH in water are added to asolution of 130 mg (0.22 mmol) of methyl4-{[(2-{2-[(4-cyclohexylbenzyl)oxy]-4-fluorophenyl}ethyl)(5-methoxy-5-oxo-pentyl)amino]methyl}benzoatefrom Ex. V in 5 ml of THF, and the mixture is stirred at 60° C. for 15hours. After cooling, the mixture is diluted with water and extractedwith ether. The aqueous phase is adjusted to pH 4 to 5 using 1-molarhydrochloric acid. This results in the precipitation of the product,which is filtered off, washed with water and dried. This gives 92 mg(0.16 mmol, 74% yield) of a white solid.

[0135] Melting point: >250° C.

[0136] R_(f) (ethyl acetate/methanol 7:3): 0.20.

[0137]¹H-NMR (300 MHz, DMSO-d₆, δ/ppm): 12.31 (2H, broad), 7.83 (2H, d),7.32 (2H, d), 7.27 (2H, d), 7.18 (2H, d), 7.12 (1H, dd), 6.90 (1H, dd),6.66 (1H, dt), 4.98 (2H, s), 3.59 (2H, s), 2.72-2.65 (2H, m), 2.59-2.51(2H, m), 2.48 (1H, m, partially obscured by DMSO), 2.47-2.68 (2H, m),2.10 (2H, t), 1.80-1.67 (5H, m), 1.44-1.19 (9H, m).

[0138] MS (ESI): 562 (M+H⁺).

[0139] The following compounds were obtained in an analogous manner:¹H-NMR spectrum: Ex. Formula δ[ppm] (DMSO-d₆) 2 (from 2- hydroxy-5-fluoroben- zaldehyde)

12.39 (2H, broad), 7.83(2H, d), 7.33-7.15 (6H, m), 7.02-6.95 (3H, m),4.96(2H, s), 3.60 (2H, s), 2.78-2.69 (2H, m), 2.63-2.57 (2H, m),2.48(1H, m, partially obscured by DMSO), 2.45-2.38 (2H, m), 2.16-2.07 #(2H, m), 1.80-1.67(5H, m), 1.46-1.21(9H,m). (200MHz) 3 (from 2-hydroxy-5- chloroben- zaldehyde and 4-(4- trifluorom- ethylphen-yl)benzyl chloride)

12.33(2H, broad), 7.90-7.78 (6H, m), 7.71(2H, d), 7.47 (2H, d), 7.32(2H,d), 7.20 (2H, dd), 7.06-7.01(1H, m), 5.10(2H, s), 3.62(2H, s), #2.80-2.71(2H, m), 2.67-2.58 (2H, m), 2.46-2.39(2H, m), 2.15-2.05(2H, m),1.44-1.37 (4H, m). (200 MHz) 4 (from 2- hydroxy-5- chloroben- zaldehydeand 4-(4- trifluorom- ethylphen- yl)benzyl chloride)

12.37(2H, broad), 7.82(2H, d), 7.58(4H, d), 7.39(2H, d), 7.32(2H, d),7.23-7.18(2H, m), 7.06-7.00(3H, m), 5.07 (2H, s), 3.80(3H, s), 3.61 #(2H, s), 2.78-2.70(2H, m), 2.64-2.57(2H, m), 2.46-2.39 (2H, m),2.16-2.07(2H, m), 1.44-1.37(4H, m). (200 MHz) 5 (from 2- hydroxy-5-chloroben- zaldehyde)

7.81(2H, d), 7.30-7.22(4H, m), 7.21-7.16(4H, m), 7.02 (2H, d), 4.97(2H,s), 3.57 (2H, s), 2.71(2H, dd), 2.57 (2H, dd), 2.5(1H, obscured byDMSO), 2.40(2H, dd narrow), 2.10(2H, dd narrow), 1.80-1.63(5H, m), #1.42-1.19(9H, m). (300 MHz) 6 (from 2- hydroxy-5- chloroben- zaldehydeand 4-(4- chlorophe- nyl)benzyl chloride)

12.33(2H, broad), 7.82(2H, d), 7.67(2H, d), 7.64(2H, d), 7.51(2H, d),7.43(2H, d), 7.31(2H, d), 7.22-7.20(2H, m), 7.05-7.02(1H, m), 5.08 (2H,s), 3.61(2H, s), 2.76 # (2H, dd), 2.60(2H, dd), 2.42 (2H, pseudo-t),2.10(2H, pseudo-t), 1.43-1.37(4H, m). (300 MHz) 7 (from 2- hydroxy-5-chloroben- zaldehyde and 4-(4- fluorophe- nyl)benzyl chloride)

12.29(2H, broad), 7.82(2H, d), 7.70-7.66(2H, m), 7.62 (2H, d), 7.42(2H,d), 7.31-7.27(4H, m), 7.21-7.19(2H, m), 7.03(1H, d), 5.07(2H, s),3.61(2H, s), 2.74(2H, dd), # 2.60(2H, dd), 2.42(2H, pseudo-t), 2.09(2H,pseudo t), 1.42-1.36(4H, m). (300 MHz) 8 (from 2- hydroxy-5- chloroben-zaldehyde and 4-(4- cyanophe- nyl)benzyl chloride)

7.93-7.88(4H, m), 7.81(2H, d), 7.72(2H, d), 7.47(2H, d), 7.31(2H, d),7.22-7.18(2H, m), 7.03-7.00(1H, m), 5.09 (2H, s), 3.61(2H, s), 2.73 #(2H, dd), 2.61(2H, dd), 2.42 (2H, pseudo-t), 2.09(2H, pseudo-t),1.43-1.37(4H, m). (200 MHz) 9 (from 2- hydroxy-5- chloroben- zaldehydeand 4-(4- methoxy- phenyl)-2- chloroben- zyl chloride)

12.36(2H, broad), 7.79(2H, d), 7.72(1H, d narrow), 7.63 (2H, d),7.57-7.50(2H, m), 7.30-7.21(4H, m), 7.09-7.00 (3H, m), 5.09(2H, s), 3.81# (3H, s), 3.57(2H, s), 2.72 (2H, dd), 2.61(2H, dd), 2.38 (2H,pseudo-t), 2.07(2H, psuedo-t), 1.37(4H). (200 MHz)

[0140] Ex. 10:4-{[(4-carboxybutyl)(2-{2-[(4-cyclohexylbenzyl)oxy]-4-fluorophenyl]ethyl)amino]methyl}benzoicAcid Hydrochloride

[0141] 0.5 ml (2 mmol) of a 4-molar solution of HCl in dioxane is addedto a solution of 220 mg (0.43 mmol) of4-{[(4-carboxybutyl)(2-{2-[(4-cyclohexylbenzyl)oxy]-4-fluorophenyl]ethyl)amino]methyl]benzoicacid from Ex. 1 in 0.2 ml of dioxane, and the mixture is stirred at 60°C. for 1 h. The mixture is then concentrated by evaporation and theresulting colorless oil is triturated repeatedly with diethyl ether. Theresulting crystals are filtered and dried.

[0142] Melting point: >250° C.

[0143]¹H-NMR 8[ppm) DMSO-d₆:12.60 (2H, broad), 10.44 (1H, s broad), 7.97(2H, d), 7.69 (2H, d), 7.32 (2H, d), 7.27-7.19 (3H, m), 7.01 (1H, dd),6.74 (1H, dt), 5.04 (2H, s), 4.38 (2H, broad), 3.15-2.92 (6H, m), 2.46(1H, m, partially obscured by DMSO), 2.14 (2H, t), 1.78-1.61 (6H, m),1.46-1.21 (8H, m). (300 MHz)

[0144] The following compound was obtained in an analogous manner:¹H-NMR spectrum: Ex. Formula δ[ppm] (DMSO-d₆) 11 (from 2)

13.00(1H, broad), 12.32(1H, broad), 10.43(1H, s broad), 7.98(2H, d),7.69(2H, d), 7.31(2H, d), 7.20-7.07(5H, m), 5.02(2H, s), 4.39(2H,broad), 3.18-2.97(6H, m), 2.44(1H, m, partially obscured by DMSO), 2.17(2H, dd), 1.79-1.61(6H, m), # 1.43-1.22(8H, m). (300MHz)

1. A compound of the general formula (I)

where R¹ is located in the meta- or para-position to the ethylaminoradical and represents halogen; R² represents H or halogen; R³represents H or halogen; R⁴ represents C₃₋₈-cycloalkyl or phenyl, wherethe phenyl radical may additionally carry a substituent from the groupconsisting of halogen, CN, OMe, CF₃; with the proviso that R⁴ may notrepresent phenyl which carries a substituent CF₃ or OMe in thepara-position to the point of attachment if, simultaneously, R¹ is inthe meta-position to the ethylamino radical and represents F and R² andR³ each represent H; and its salts, isomers and hydrates.
 2. A compoundas claimed in claim 1, characterized in that R¹ is located in the meta-or para-position to the ethylamino radical and represents F, Cl or Br;R² represents H or F or Cl; R³ represents H; R⁴ represents cyclohexyl orphenyl, where the phenyl radical may additionally carry a substituentfrom the group consisting of F, Cl, Br, CN, OMe, CF₃; with the provisothat R⁴ may not represent phenyl which carries a substituent CF₃ or OMein the para-position to the point of attachment if, simultaneously, R¹is in the meta-position to the ethylamino radical and represents F andR² and R³ each represent H; and its salts, isomers and hydrates.
 3. Acompound as claimed in claim 1, characterized in that R¹ is located inthe meta-position to the ethylamino radical and represents Cl; R²represents H or Cl; R³ represents H; R⁴ represents cyclohexyl or phenyl,where the phenyl radical may additionally carry a substituent from thegroup consisting of F, Cl, CN, OMe, CF₃; and its salts, isomers andhydrates.
 4. A compound as claimed in claim 1, characterized in that R¹is located in the para-position to the ethylamino radical and representsCl or F; R² represents H or Cl; R³ represents H; R⁴ representscyclohexyl or phenyl, where the phenyl radical may additionally carry asubstituent from the group consisting of F, Cl, CN, OMe, CF₃; and itssalts, isomers and hydrates.
 5. A process for preparing compounds of thegeneral formula (I), characterized in that compounds of the formula (II)

in which R¹, R², R³ and R⁴ are as defined in claim 1, are reacted with aC₁₋₆-alkyl 4-formylbenzoate in an organic solvent with heating and withsimultaneous or subsequent addition of a reducing agent to givecompounds of the formula (III)

where R¹, R², R³ and R⁴ are as defined above and Q represents aC₁₋₆-alkyl radical, then reacted with a C₁₋₆-alkyl ω-halovalerate in anorganic solvent in the presence of a base with heating to give compoundsof the formula (IV)

where R¹, R², R³ and R⁴ and Q are as defined above and Q′ represents aC₁₋₆-alkyl radical, and the compounds of the formula (IV) are thenhydrolyzed under alkaline conditions to give the compounds of theformula (I).
 6. A compound as claimed in any of the preceding claims fortreating diseases.
 7. A medicament, comprising at least one compound ofthe general formula (I) as claimed in any of the preceding claims. 8.The use of compounds of the formula (I) as claimed in any of thepreceding claims for preparing a medicament for treating cardiovasculardisorders.
 9. The use of compounds of the general formula (I) as claimedin any of the preceding claims for preparing medicaments for treatingangina pectoris, ischemias and heart failure.
 10. The use of compoundsof the general formula (I) as claimed in any of the preceding claims forpreparing medicaments for treating hypertension, thromboembolicdisorders, arteriosclerosis and venous diseases.
 11. The use ofcompounds of the general formula (I) as claimed in any of the precedingclaims for preparing medicaments for treating fibrotic disorders. 12.The use as claimed in claim 9, characterized in that the fibroticdisorder is fibrosis of the liver.